Attenuators

ABSTRACT

Methods for detecting nucleic acid sequences, where attenuator oligonucleotides are provided to reduce the number of detection products resulting from highly abundant sequences.

CROSS-REFERENCE

This application is a division of U.S. application Ser. No. 14/480,525,filed on Sep. 8, 2014, which is incorporated herein by reference in itsentirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grants1R43HG007339-01 and 5R43HG007339-02 awarded by the National Institutesof Health. The government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created Mar. 21, 2018, isnamed “42722701401_SL.txt” and is 2,829 bytes in size.

BACKGROUND OF THE INVENTION

This invention relates to molecular biology, and more particularly tomethods for detecting nucleic acid sequences where certain sequences ina sample are highly abundant.

BRIEF SUMMARY OF THE INVENTION

This invention provides methods for detecting target nucleic acidsequences of interest in a sample. In a typical ligation assay, thesample is contacted with a pool of detector oligonucleotides, where apair of detector oligonucleotides is provided for each target sequence:a downstream detector (DD) and an upstream detector (UD). A downstreamdetector can have a portion (DR′) that is complementary to a region ofthe target sequence designated as a downstream region (DR). An upstreamdetector can have a portion (UR′) that is complementary to a region ofthe target sequence designated as the upstream region (UR).

The detectors are allowed to hybridize to nucleic acids in the sample.When downstream and upstream detectors both hybridize to thecorresponding regions of the same target sequence, and the DR and UR aredirectly adjacent, the detector oligos can be ligated. Where the DR andUR are separated by one or more nucleotides, the DR can be extendedprior to ligation to the UR. Formation of a ligation product thus servesas evidence that the target sequence was present in the sample. In someassay formats, the ligation product can be amplified with primers (e.g.P1, P2) where the detector oligos have corresponding primerhybridization sequences (e.g. P1, P2′), or the complements thereof. Theligation product (or its amplicons) can be detected by methods such aslabeling for detection on an array, qPCR, or sequencing.

Where certain target sequences may be highly abundant in a sample,however, it can be desirable to detect the presence of those highlyabundant target sequences (HATs), while attenuating the overall numberof HAT-related ligation or amplification products to be detected, inorder to facilitate the detection of the other target sequences ofinterest in the sample. Accordingly, this invention provides attenuatoroligonucleotides. Some attenuators are provided that can replace one orboth detectors for a HAT. Other attenuators can be contacted with thesample in competition with the detectors. This invention also providessets of attenuator oligonucleotides, which can be specific for cell ortissue types, and kits containing such attenuators.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a representative ligation assay for detection oftarget nucleic acid sequences.

FIG. 2 depicts various oligonucleotides that attenuate primarily beforeand during hybridization. When an attenuator oligonucleotide is depictedwith an optional sequence (sometimes a “tail”), this is depicted by adotted line.

FIG. 3 depicts various oligonucleotides that attenuate primarily duringligation or extension.

FIG. 4 depicts various oligonucleotides that attenuate primarily duringamplification.

FIG. 5 shows the attenuated detection of the target sequence GAPDH_2when using various combinations of mutated downstream attenuators andmutated upstream attenuators.

FIG. 6 shows the attenuated detection of GAPDH_2 using bridgeattenuators, while showing the enhanced detection of selectedless-abundant target sequences.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides methods for detecting target nucleic acidsequences of interest in a sample. The sample can be any substance whereit is desired to detect whether a target sequence of a nucleic acid ispresent. Such samples can be from living or dead organisms, or fromartificially created or environmental samples. The samples can be in theform of tissue samples, cell samples, or samples that are cell-free. Thesamples can be provided in liquid phase, such as cell-free homogenatesor liquid media from tissue cultures, or in solid phase, such as whenthe sample is mounted on a slide or in the form of formalin-fixedparaffin-embedded (FFPE) tissue or cells.

The target nucleic acid sequence of interest to be detected in a samplecan be a sequence or a subsequence from DNA, such as nuclear ormitochondrial DNA, or cDNA that is reverse transcribed from RNA in thesample. The sequence of interest can also be from RNA, such as mRNA,rRNA, tRNA, miRNA, siRNAs, antisense RNAs, or long noncoding RNAs. Moregenerally, the sequences of interest can be selected from anycombination of sequences or subsequences in the genome or transcriptomeof a species or an environment.

Highly Abundant Target Sequences of Interest (HATs)

In cases where there is more than one target sequence of interest in agiven sample, it is likely that they will be present in differentamounts. Moreover, the amount of a target sequence can vary amongsimilar samples. Ideally, a detection assay will have sufficient dynamicrange to measure the presence of the different target sequencesquantitatively in a single experiment. For some types of samples,however, the range of abundance for various sequences of interest canspan several orders of magnitude. For example, when profiling the RNAexpression products of a cell, individual sequences of particularinterest may be present in very few copies, while others are highlyabundant target sequences (HATs). The HATs can be present in a sample insuch large numbers that they may diminish the ability of a method todetect the presence of less abundant target sequences.

Depending on the cell or tissue type, such highly abundant HATs caninclude sequences encoding what are generally referred to ashousekeeping genes. Examples of HATs include sequences that encode allor a portion of myoglobins, actins, tubulins, ubiquitins, heat-shockproteins (HSPs), ribosomal proteins, ribosomal RNAs (rRNAs), micro-RNAs(miRNAs), or small nuclear RNAs (snRNAs). Other examples of HATs canencode all or a portion of cytochrome c, glyceraldehyde 3-phosphatedehydrogenase (GAPDH), ribosomal protein L7 (RPL7), ribosomal protein S6(rpS6), snRNA RNUs, phosphoglycerokinase (PGK), tyrosine3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta(YWHAZ), beta-actin, or beta-tubulin. Further examples include sequencesencoding all or a portion of alpha-2-microglobulin, vimentin, andfibronectins. Yet other examples of HATs encode all or part of acytochrome such as mitochondrially encoded cytochrome b (MT-CYB), outermitochondrial membrane cytochrome b5 type B, microsomal cytochrome b5type A (ACYB5A), and ascorbate-dependent cytochrome b3 (CYBASC3).

Because which sequences are highly abundant can differ from one sampletype to another, such as between different tissues or cell types,certain target sequences can be designated as a predetermined set ofpotential HATs based on a search of the literature for that type ofsample, or can be determined by performing preliminary assays todetermine the more abundant sequences in the sample type. For somesample types, the number of HATs in a predetermined set can range in anycombination of upper and lower limits of 1, 2, 5, 10, 20, 50, 100, 200,500, 1000, 2000, 5000, and 10,000. For example, a useful predeterminedset can have 1-10 HATs, 5-20 HATs, or 100-5000 HATs. Likewise, thenumber of HATs in a predetermined set can be expressed as a percentageof the total number of target sequences of interest, ranging in anycombination of upper and lower limits of 0.1%, 0.2%, 0.5%, 1%, 2%, 5%,10%, 20%, 25%, 30%, 35%, 40%, 45%, and 50%.

Ligation Assays

While many methods can be used to detect the presence of a targetsequence, a representative method is a ligation assay, such as inExample 1 and illustrated schematically in FIG. 1. In a typical ligationassay, the sample is contacted with a pool of detector oligonucleotides(“detectors”). For each target sequence of interest, a pair of detectorsis provided: a downstream detector (DD) and an upstream detector (UD). Adownstream detector can have a portion (DR′) that is complementary to aregion of the target sequence designated as a downstream region (DR). Anupstream oligo can have a portion (UR′) that is complementary to aregion of the target sequence designated as the upstream region (UR).Here, the terms “downstream” and “upstream” are used relative to the5′-to-3′ direction of transcription when the target sequence is an mRNA.The DR and UR of a target sequence are typically subsequences of theentire target sequence of interest, and an individual target sequencecan have more than one set of DRs and URs, which can be selected by theuser to optimize the performance of the assay. Multiple sets of DRs andURs can provide multiple measurements of the same target sequence or ofdifferent portions of the target sequence, such as different exons orexon junctions, or provide measurement of a portion of sequence that isnot mutated versus a portion of sequence that may harbor a mutation. Insome cases, the DR and UR are directly adjacent; in other cases, theycan be separated by one or more nucleotide positions on the targetsequence. In other cases, a portion of the DD can overlap with the URsequence to which the UD hybridizes so that after hybridization of boththe UD and the DD, there is an overhang sequence of 1, 2, 3, or morebases.

The DD or UD, or both, can contain a barcode sequence. For example, auseful barcode sequence can uniquely identify the specific gene ortarget sequence, or a group of select genes or target sequences withinthe sample that are being measured. Such sequences can be positionedbetween the UR′ and P2′ sequence, and/or between the DR′ and P1sequence, so they are amplified when using flanking primers.

In a ligation assay, the pool of detector oligos is contacted with thesample. As shown in FIG. 1, the DR′ of the DD and the UR′ of the UD foreach target sequence are allowed to hybridize (a) to the correspondingDR and UR of the target sequence, if present in the sample, serving as atemplate.

When the DR and UR of a target sequence are directly adjacent, thedetector oligos can be ligated (b): thus formation of a ligation productserves as evidence that the target sequence (DR+UR) was present in thesample. The ligation reaction can occur by chemical ligation or by usinga ligase. A variety of nick-repairing ligases are commercially availableto catalyze the formation of a phosphodiester bond between adjacentsingle-stranded polynucleotides when hybridized to anothersingle-stranded template. An example is bacteriophage T4 DNA ligase,which uses ATP as a co-factor. The ATP can be supplied during the ligasereaction. In other reactions, the ligase can be pre-adenylated. In otherreactions, the UD must be pre-adenylated at the 5′ end, as with a 5′ AppDNA/RNA ligase. The UD in a typical reaction will have a 5′-phosphate tofacilitate ligation to the DD, although this is not necessary, dependingon the selection of ligase and ligation conditions. Where a 5′-phosphateon the DD is required for efficient ligation, using a comparableoligonucleotide without 5′-phosphorylation can be used to inhibitligation.

In cases where DR and UR are separated by one or more nucleotidepositions on the target sequence template, an extension step (b1) can beperformed, as shown in FIG. 1, followed by the ligation step (b2).Ligation can also be preceded by a cleavage step, such as by a nuclease,to remove any overhangs. A useful enzyme is a Flap endonuclease (FEN),such as Fen-1.

Where the ligation assay proceeds directly to a detection step, eitheror both detectors can be designed to be labeled appropriately fordetection. For example, the detector can be labeled with a color orfluorescent dye, latex bead, quantum dots, or nanodots. The label canalso take the form of an additional nucleotide sequence that serves toenable detection and identification, such as a barcode sequence.

In some embodiments, the hybridization, ligation, or extension steps canbe performed while the target sequence is in situ. This can beparticularly useful, for example, when the sample is on histologicalslide, so that the ligation is known to occur at a recordable locationand can be compared to similar reactions at other locations on theslide. In a particular embodiment, the ligation products can be elutedfrom the sample in situ for collection and further processing,preferably eluting from small areas to preserve the location informationof the ligation reaction products.

In some assay formats, the ligation products can be (c) amplified tofacilitate detection. As illustrated in FIG. 1, the detectors can haveadditional sequences (“tails”) including primer hybridization sequences(e.g. P1, P2′) or complements thereof, that serve as amplificationsequences, so that after ligation, the ligation product can be amplifiedwith a pair of amplification primers (P1, P2). An exemplary downstreamamplification sequence (P1) is 5′-CAAGCAGAAGACGGCATACGAG-3′ (SEQ ID NO:10), which can be used with a primer having the same sequence (P1). Anexemplary upstream amplification sequence (P2′) is5′-ATCTCGGTGGTCGCCGTATCATT-3′ (SEQ ID NO: 11), which can be used withprimer P2 (shown in 3′-5′ orientation): 3′-TAGAGCCACCAGCGGCATAGTAA-5′(SEQ ID NO: 12).

If desired, the amplification primer can incorporate a barcode sequence,for example a barcode sequence that uniquely identifies the sample in amulti-sample experiment. The barcode sequence can be incorporated intothe primer, such as 3′ to the amplification sequence, so that thebarcode becomes part of the amplified strand. In other instances, theamplification sequence of the primer can be extended by an additionalsequence to provide a primer hybridization sequence that can be used foruse in subsequent sequencing steps. The barcode may also be interposedbetween the amplification sequence, and if desired, the extendedamplification sequence, and another sequence that can be used forcapture, such as capture onto a surface as part of a sequencing process,and/or for yet another primer hybridization sequence that is used forsequencing. In each case the barcode will be amplified with the rest ofthe detector sequences, for instance forming a single amplified,elongated molecule that contains sequencing primer hybridizationsequences, sample barcode, and a gene-specific sequence, which mayinclude a gene-specific barcode as well as sequence or complement to thesequence of the target gene. In the case where the targeted oligo is acDNA, a gene-specific sequence or a sample specific sequence can beadded as part of the primer used for reverse transcription, and be apart of the sequence targeted by the UD and DD.

In other instances, methods known in the art can be used to amplify theligated DD and UD sequences, such as by repetitive cycles of (1)ligation, (2) heating to melt off the ligated product, (3) cooling topermit hybridization of DD and UD to the target, (4) ligation, thenrepeating the heating (2), cooling (3), and ligation (4) steps. Theseadditional amplification steps can be performed before amplificationstep (c), during which the sample barcodes and other sequences are addedto the ligated UD and DD sequence. The target of the UD and DDhybridization may also be amplified by whole transcriptome amplificationof RNA or amplification of cDNA.

The ligation product (or its amplicons) can then be detected by methodssuch as sequencing, qPCR, or labeling for detection on an array.Depending on the detection method, the skilled user will be able tomodify the design of the detectors and amplification primers to includefunctional features that are appropriate, such as for bridgeamplification on a sequencing flow cell.

Attenuators

In the context of the assays described above, this invention disclosesvarious attenuator oligonucleotides (“attenuators”) that can be used toattenuate the overall number of HAT-related ligation or amplificationproducts to be detected. Some attenuators are provided that can replaceone or both of the detectors for a HAT to provide positive detection ofthe HAT in the sample, but at a lower level of signal. These and otherattenuators can also be added to the ligation reaction to attenuate thesignal for the HATs. For purposes of discussion, the various attenuatorscan be also grouped as interacting with the detectors and targetsequences at different stages of the assay. However, the Applicant doesnot wish to be bound by any proposed mechanisms of action as long asattenuation of detection is achieved.

Attenuators can hybridize competitively with part or all of a DR and/orUR of a HAT. As shown in FIG. 2, attenuator 21 can hybridize to aportion of a DR, reducing access of a corresponding DD to the same DR.Similarly, attenuator 22 can hybridize competitively to a portion of anUR. An attenuator 23 or 24 can also have a portion that is complementaryto regions of the HAT beyond the DR or UR. Because the length of suchextended attenuators may be more comparable to the length of a DD or UDin the assay, their binding properties may be more thermodynamicallysimilar to a DD or UD. Thus, the overall length of an attenuator, notjust the ratio of a specific attenuator concentration to the DD or UDconcentration in the hybridization cocktail, can be tuned to provide thedesired level of attenuation for a particular HAT. As with allattenuators disclosed herein, the length and sequence of theoligonucleotide can be tuned for desired properties such as specificity,and annealing and melting temperatures. For example, an attenuator maybe tuned to increase or decrease the number of C:G pairs formed duringhybridization steps of the assay.

A bridge attenuator 25, which has portions that are complementary tosubsequences of the DR or UR, may also compete effectively with both DDsand UDs for binding to the DR and UR. A full bridge attenuator 26 blocksthe DR and UR of a HAT from hybridizing with either or both of a DD orUD, and its effectiveness is illustrated in Example 3.

A class of circularizing attenuator is provided that has hybridizationsequences connected via a linking sequence so that when it hybridizes toa portion of the target sequence, it circularizes using the targetsequence as a splint. For example in FIG. 2, attenuator 27 has a DR′ andUR′ connected by a flexible linker. The attenuator can also have variouscombinations of sequences that hybridize

-   -   to at least part of the DR and the UR (as in 28, for example);    -   to a portion of a DR, in addition to adjacent downstream        sequence of the target sequence (as in 28);    -   to a portion of an UR, in addition to adjacent upstream sequence        of the target sequence;    -   to two different portions of the DR (as in 29) or UR; and/or    -   to a portion of either or both of the DR or UR with one end of        the circularizing attenuator, and to a region of the sample that        is separated by one or more nucleotides from the DR or the UR of        the target sequence (as in 30);        as long as the hybridization of the circularizing attenuator        reduces the number of UD or DDs hybridizing or ligating at the        target sequence of a HAT. The 5′ ends of the circularizing        attenuators may be phosphorylated to facilitate ligation as        appropriate to the ligation enzyme and conditions used.

As single molecules, these circularizing molecules can have superiorhybridization characteristics compared to two separate attenuatorshaving similar complementary regions. In contrast to a molecularinversion (padlock) probe, however, the oligonucleotide does not serve adetection function, but to attenuate the formation of HAT-relatedproducts to be detected.

Attenuators are provided that have a group that blocks effectiveligation between a DD and an UD during the assay. For example, in FIG.3, an attenuator 31 hybridized to a DR of a HAT can be similar to a DD,except having a blocking group at the 3′ end to prevent ligation to anUD hybridized to the same HAT. Likewise, an attenuator 32 similar to anUD can be provided with a blocking group at the 5′ end to preventligation between the DD and the attenuator. Useful blocking groupsinclude nucleotides with modified bases (as discussed below) and spacergroups that prevent ligation with the ligase or chemical ligation methodbeing used.

Examples of blocking groups at the 3′ end for the UD include a2′,3′-dideoxynucleotide, 3′-propyl, 3′-dehydroxylation, 3′-amination,and an inverted (3′-3′) linkage.

Examples of blocking groups at the 5′ end for the DD include5′-amination, 5′-adenylation, 5′-hexynyl, and an inverted (5′-5′)linkage.

For ligases that require a double-stranded substrate, aligation-blocking group can be a portion of nonhybridizing sequence. Forexample, an attenuator 33 that binds to a DR will present asingle-stranded portion that will not ligate (or at least ligate atlower efficiency) than a comparable DD. Similarly, an attenuator 34 willligate less efficiently, if at all, to a DD hybridized to the same HAT.Yet another attenuator 35 can have a nonhybridizing portion that forms asecondary structure, such as hairpin loop, to reduce ligation.

An embodiment of attenuator oligonucleotide that can replace a detectorcan have a portion partially complementary to the downstream or upstreamregion of the HAT. A particular embodiment is when the attenuator has asequence similar to a detector, but has one or more mutated positions.Examples of such mutated attenuators are described in Example 2. Somemutated DDs have one (36), two (37, 38), or three mismatches (relativeto the DR template) at positions at (36, 37) or near (38) the 3′ end.Some mutated UDs have one, two, or three mismatches (relative to the URtemplate) at positions at or near the 5′ end. A mutated DD can be usedin combination with an unmutated (“wildtype”) UD; a mutated UD can beused with a wildtype DD; or various mutated upstream and downstreamdetectors can be combined, depending on the degree of attenuationdesired. Moreover, a mutated attenuator for a HAT can also be usefulwhen provided during hybridization in addition to the wildtype detectorsfor the same HAT.

Another embodiment of attenuator reduces the number of ligation productsto be detected by being more difficult to separate from the targetsequence than comparable detectors. For example, attenuators may bedesigned to have higher melting temperatures so that fewer detectors areable to compete for the same target sequences. Attenuators can also bedesigned so that when hybridized to the target sequence with a detectoror a second attenuator, the ligation product is more likely to remainhybridized to the target than the ligated pairs of detectors. This canbe achieved by modifying the attenuator to cross-link to the target viaoligo-directed cross-linking or by photo-activated cross-linking.

Yet another embodiment of an attenuator contains a modification or asegment that renders the attenuator or a ligation product more difficultor impossible to amplify when an amplification step is included in theassay.

An example of such an attenuator has a full-length DR′ or UR′, but lacksany sequence that can serve as a primer sequence for the primers beingused during the amplification step. In FIG. 4, the upstream attenuator41 has a 5′ tail (X) that is different from the P1 sequence that wouldenable amplification by a P1 primer. Similarly, downstream attenuator 42has a non-amplifiable sequence (Y) as a 3′ tail that is different fromsequence P2′. Where it is desirable for the tails of attenuators to havea complete P1 or P2′ sequence, the tails can be extended to incorporatea reverse complementary sequence, forming a hairpin loop, to reduce theamplification of ligation products. In other cases, a self-cleavingribozyme sequence can be incorporated into an attenuator tail to renderthe ligation product unamplifiable.

Attenuators for reduced amplifiability 43 and 45 incorporate achemically modified segment (44, 46) that, while allowing ligation to adetector or a second attenuator, is more difficult to amplify than theligation product of two wildtype detectors. The selection of themodification will depend on the particular polymerase being used in theamplification step. For example, Pfu polymerase stalls when a DNAtemplate contains deoxyUridine (dU) or 2′-deoxylnosine. Another exampleis a segment that has a 1′,2′-dideoxyribose sugar, resulting in anabasic site. Other segments can include a modified nucleotide such asdideoxy nucleotides, deoxyUridine (dU), 5-methylCytosine (5mC),5-hydroxymethylCytosine (5hmC), 5-formylCytosine (5fC), and5-carboxylCytosine (5caC), and Inosine. Yet other segments can havenucleotides having modified bases such as 2,6-diaminopurine,2-aminopurine, 2-fluro bases, 5-bromoUracil, or 5-nitroindole.

Other nonamplifiable attenuators include oligonucleotides having asugar-phosphate backbone that has a 3′-3′ or 5′-5′ linkage inversion ora peptide nucleic acid (PNA) backbone.

Another embodiment of useful attenuators contains one or modificationsthat can be cleaved by treatment after the ligation or optionalamplification step. For example, an attenuator can have a dU located sothat it will not interfere with hybridization or ligation steps. Afterligation, however, products incorporating the dU attenuator can then becleaved by dU-specific enzymes, such as uracil-DNA glycosylase followedby endonuclease VIII. The cleavage products are thereby renderedunsuitable for amplification or for detection steps that rely onfull-length ligation products. An attenuator particularly vulnerable todegradation is a oligonucleotide that contains one or more RNAnucleotides: the resulting ligation product is susceptible to thepresence of high pH or Mg′ ions, which can occur during amplificationreactions such as PCR, effectively preventing RNA-containing ligationproducts from providing a useful measurement of a target sequence in anassay.

Another approach is to incorporate into an attenuator a selectivelycleavable site so that the attenuator can be cleaved without affectingthe other components of the assay. For attenuators 43 and 45, such asite can be placed at 44 or 46, for example. A selectively cleavablesite can be a restriction enzyme cleavage site that is not present inthe wildtype detectors or in the target sequences of interest to bedetected. Treatment with the restriction enzyme selectively cleaves theattenuator so that it does not form a detectable product. If anamplification step is used, the restriction treatment can be performedbefore amplification to reduce amplification of HAT-derived ligationproducts, or after amplification to cleave HAT-derived amplificationproducts. If sequencing is used as a detection method, the result of therestriction treatment is a reduction in the number of sequenceableproducts arising from the HAT.

The various features described above can be implemented in a singleattenuator, for example an oligonucleotide that has both a block toprevent ligation and a tail that will not be amplified. When attenuatorsare used in pairs for a target sequence, they can have differentfeatures, such as a downstream attenuator with two mutated nucleotides,and a downstream attenuator with a tail that forms a hairpin loop.

Attenuators can also be used at more than one step of the method. Forexample, if greater attenuation of a particular HAT is desired, morethan one attenuator can be provided, such as two or more taillessversions of attenuators 21-25 in a single hybridization reaction. Inaddition, oligonucleotides can be spiked into the hybridization oramplification steps, where the oligonucleotides contain DR or UR, orsubsequences thereof, to compete with the DR and UR target sequencespresent in the sample.

If it is desired to attenuate three HATs of a sample independently, itmay be desirable to attenuate a first target sequence at thehybridization step, attenuate a second target sequence at the ligationstep, and attenuate a third target sequence at the amplification step,controlling the degree of attenuation at each step.

If even further attenuation of detectable HAT products is desired, thesample can be pre-treated to remove HAT target sequences prior to step(a), for example by using immobilizable beads or other solid phase thatcontain oligonucleotides that are specifically complementary to the HATtarget sequences. Beads to remove rRNA and globin sequences arecommercially available. If a capture sequence on a solid phase surfacehybridizes to the UR and/or DR, or a portion of either, then includingthe capture sequence with the DD and UD at a predetermined ratio candeplete a portion of the HAT. Similarly, an oligo that targets the URand/or DR or a portion of one or both—and that in turn can be capturedonto a surface through a second sequence—can be used to compete with theDD and/or UD. Then, the HAT—to which the competitive, capturableoligonucleotide is hybridized—can be removed from the sample. Oneskilled in the art will see that there are many combinations that can beused for selective capture and depletion of HATs in a sample.

The selection of the attenuators used in an experiment will depend onthe degree of attenuation desired for the particular HAT in a sample. Ifthe target sequences are to be detected after ligation, the desiredresult will be a decreased number of HAT-related ligation products to bedetected. If an amplification step is included, the attenuation canresult in fewer HAT-related amplification products to be generated anddetected. The net result is to optimize the utilization of assay anddetection resources among HATs and the other target sequences ofinterest. The use of attenuators is particularly effective when therelative number of amplification products for other target sequences ofinterest is maintained, and preferably enhanced, as demonstrated inExample 3.

The attenuators of the invention can provide attenuation of detected HATproducts in a range of any combination of upper and lower limits of 20%,30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%,99%, 99.5%, 99.7%, 99.9%, 99.95%, or 99.99% or more, compared toproducts detected by wildtype HAT detectors.

This invention also provides sets of attenuator oligonucleotidesdescribed above. The set can be specific for a sample type, such as acell or tissue type, that has a plurality of HATs. This inventionfurther provides kits containing the attenuators. The kits and methodsof the invention can include attenuators in a range of any combinationof upper and lower limits of 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000,2000, 5000, 10,000, or more HATs. In another aspect, the number of HATsfor which attenuators are provided can be in a range of any combinationof upper and lower limits of 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.5%, 1%,2%, 5%, 10%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, or more of the totalnumber of target sequences for a sample.

The kits can be provided with detectors for target sequences ofinterest. The kits can also have eluent solutions suitable for removingoligonucleotides, such as ligated oligonucleotides, from a tissue samplefor further analysis. The kits can further have amplification primerssuitable for use with the detectors of the kit.

EXAMPLES Example 1: Representative Ligation Assay

A representative method is provided to illustrate ligation assayswithout the attenuators of the invention. Here, over 100 RNA expressionproducts were detected in a sample of cells using a multiplex assayformat. For each expression product, the assay was designed to detectone or more sequences of interest within the full sequence of theproduct. For example, in human cells, a GAPDH gene encodes the enzymeglyceraldehyde 3-phosphate dehydrogenase; three different sequences ofinterest within the RNA transcript of the GAPDH gene were independentlydetected. One such RNA sequence, identified here as GAPDH_2, was

(SEQ ID NO: 1) 5′-CGACCACUUUGUCAAGCUCAUUUCCUGGUAUGACAACGAAUUUGGCU ACA-3′where a 5′ end was designated “upstream” (underlined) and the 3′ end wasdesignated “downstream” for the direction of transcription andtranslation. The same GAPDH_2 sequence can be shown in the 3′-to-5′direction for later convenience of discussion:

(SEQ ID NO: 1) 3′-ACAUCGGUUUAAGCAACAGUAUGGUCCUUUACUCGAACUGUUUCACC AGC-5′A downstream region (DR) was defined as the downstream 25 bases ofGAPDH_2:

(SEQ ID NO: 2) 3′-ACAUCGGUUUAAGCAACAGUAUGGU-5′which has a complementary DNA sequence of DR′:

(SEQ ID NO: 3) 5′-TGTAGCCAAATTCGTTGTCATACCA-3′The upstream region (UR) was defined as the upstream 25 bases ofGAPDH_2:

(SEQ ID NO: 4) 3′-CCUUUACUCGAACUGUUUCACCAGC-5′which has a complementary DNA sequence of UR′:

(SEQ ID NO: 5) 5′-GGAAATGAGCTTGACAAAGTGGTCG-3′

For GAPDH_2, a pair of detectors was designed: a downstream detector(DD) having the DR′ sequence, and an upstream detector (UD) having theUR′ sequence. Similar pairs were designed for each of the targetsequences of interest to provide a pool of detectors for the assay. Inthis example, all the upstream detectors were phosphorylated at the 5′end.

In this particular example, an amplification step was to be performedlater in the experiment using two primers, P1 and P2, so all UDs in theexperiment included a primer sequence (P1) and all URs included acomplementary primer sequence (P2′). Because amplification is notnecessary to the practice of the invention, however, the sequence of thespecific primers and primer sequences is a matter of selection to suitthe particular amplification method, if used.

At least 10 ng of RNA isolated from human kidney or liver cell lines wasplaced in a well of a microtiter plate for each assay experiment. Toeach well was added 20 μL of 2× Binding Cocktail, which contained 5 nMof each detector (providing a final input of 0.1 pmoles per oligo), 100nM biotinylated oligo(dT)₂₅ (SEQ ID NO: 13), and 5 μLstreptavidin-coated magnetic beads in a Wash Buffer (40 mM Tris-Cl pH7.6, 1 M NaCl, 2 mM EDTA disodium, 0.2% SDS).

The plate was heated for 10 min at 65° C. to denature the RNA, then thetemperature was ramped down over 40 min to 45° C. to allow the detectorsto anneal to the RNA sample. The plate was then transferred to amagnetic base to immobilize the beads, allowing the supernatant,containing unbound and excess detectors, to be aspirated from the wells.The beads were washed at least three times with 50 μL Wash Buffer.

To each well was added 5 Weiss units of T4 DNA ligase in 20 μL of 1×ligation buffer, as provided by the supplier. After the beads wereresuspended by pipette, the plates were incubated for 60 min at 37° C.to allow template-dependent ligation of DDs to UDs as appropriate. Afterthe ligation reaction, the beads were immobilized and washed twice with50 μL Wash Buffer. To release the ligated detectors from their RNAtargets, the beads were resuspended in 30 μL and incubated for 5 min at65° C. After incubation, the beads were immobilized, and the supernatantwas removed and transferred to a storage plate.

For the optional amplification step, 5 μL of the supernatant, containingthe ligation products, was transferred to a well of a PCR plate. Then 10μL of a PCR cocktail was added, containing 0.45 U Taq polymerase, 0.6 μMP1 primer, 0.6 μM P2 primer, 1.5 mM MgCl₂, and 200 μM dNTPs. Thethermocycler used the following program: 10 min at 94° C., followed by20 to 25 cycles of 30 sec at 94° C., 30 sec at 58° C., and 30 sec at 72°C. The amplification products were then sequenced according tomanufacturer's instructions. This representative ligation assay can bemodified by the attenuators of the invention as in the followingexamples.

Example 2: Mutated Sequence Attenuators

In this experiment, the DD and/or UD were replaced with variousattenuator oligos having one, two, or three mismatched bases. Asdiscussed in Example 1, the DR′ of the DD for GAPDH_2 had the sequence5′-TGTAGCCAAATTCGTTGTCATACCA-3′ (SEQ ID NO: 3), so that the threenucleotides at the 3′ terminus can be represented as -CCA-3′. The fullsequence can be designated as the wildtype DD. Mutated versions of theDD were prepared, each having 3′-terminal sequences as follows (mutatedbases shown in bolded lowercase):

mutated downstream attenuator 3′-terminus positions wildtype DD —CCA 0GAPDH_MM3_0b_D —CCt 1 GAPDH_MM3_1b_D —CgA 1 GAPDH_MM3_2b_D —gCA 1GAPDH_MM3_3b_D —gCt 2 GAPDH_MM3_5b_D —ggA 2 GAPDH_MM3_6b_D —Cgt 2GAPDH_MM3_4b_D —ggt 3

Similarly, the UR′ of the UD had the sequence 5′GGAAATGAGCTTGACAAAGTGGTCG-3′ (SEQ ID NO: 5), which can be designated asthe wildtype UD, with a 5′-terminal sequence of/5Phos/GGA-. In thisexample, the sequence derived from the upstream regions remainsunderlined. Attenuator versions of the UD were prepared, each having5′-terminal sequences:

mutated upstream attenuator 5′-terminus positions wildtypeUD /5Phos/GGA—0 GAPDH_MM3_0b_U /5Phos/ cGA— 1 GANDH_MM3_1b_U /5Phos/GcA— 1GAPDH_MM3_2b_U /5Phos/GGt — 1 GAPDH_MM3_3b_U /5Phos/ cGt — 2GAPDH_MM3_5b_U /5Phos/Gct— 2 GAPDH_MM3_6b_ U /5Phos/ ccA— 2GAPDH_MM3_4b_U /5Phos/ cct — 3

Combinations of the 8 DDs (wildtype and 7 mutated sequences) and 8 UDs(wildtype and 7 mutated sequences) were tested for attenuation ofligation in 64 experiments on RNA isolated from human kidney cell lines.As shown in FIG. 5, the positive control, using the wildtype DD andwildtype UD, correctly detected the presence of GAPDH_2 in the sampleRNA by generating a species that joined DR′ to UR′, and specificallycontaining the following internal sequence at the ligation junction:

(SEQ ID NO: 6) 5′-CCAGGA-3′

In each of the experiments, a DD and an UD were provided for a ligationexperiment, and the ligation products were analyzed by sequencing andcounting the number of reads containing DR′ joined to UR′, except withone of the 64 possible internal sequences formed by the junction. Forexample in one experiment, the ligation reaction was provided withdownstream attenuator GAPDH_MM3_2b_D (or “2b_D”) serving as thedownstream detector for GAPDH_2, and upstream attenuator GAPDH_MM3_2b_U(“2b_U”) serving as the upstream detector for GAPDH_2. In the presenceof GAPDH_2 sequence in the RNA sample, the pair of upstream anddownstream attenuators generated a certain number of ligation productshaving the internal sequence

(SEQ ID NO: 7) 5′-gCAGGt -3′

The formation of these ligation products was sufficient to correctlydetect the presence of GAPDH_2 in the samples, but at an attenuatedlevel (33%) compared to the comparable experiment using wildtypedetectors.

Greater attenuation was observed when using pairs of attenuators havingmore than one mutation. For example, the pairing of 5b_D and 3b_Uyielded ligation products with the internal sequence

(SEQ ID NO: 8) 5′-ggA cGt -3′which resulted in detection of GAPDH_2, but at a much reduced level ofonly 0.58% of the positive control, representing an attenuation of99.42%. No ligation products were detected when using the pair of 4b_Uand 4b_U, with three mutations at each terminus. As disclosed herein,the degree of attenuation is not easily correlated with the number orposition of mismatches. For example, the pairing of 0b_D and 0b_Uyielded an attenuated level of 7.3%, which was ten-fold higher than mostother combinations with similar attenuators. Nevertheless, whenattenuation of a HAT such as GAPDH_2 is desired, the use of mismatchedattenuators provides authentic detection of HATs without generatingundesirable numbers of ligation product.

Example 3: Bridge Attenuators

Ligation assays were performed where a bridge attenuator was added to aligation reaction, in addition to the wildtype DD and wildtype UD, at apre-determined ratio of concentration of attenuator to concentration ofwildtype DD and wildtype UD. The sample was RNA from a human kidney cellline. An attenuator specific for the DR and UR of GAPDH_2 was prepared:

(SEQ ID NO: 9) 5′-TGTAGCCAAATTCGTTGTCATACCAGGAAATGAGCTTGACAAAGTGG TCG-3′and included in a pool of 9 bridge attenuators for various attenuationtargets. The attenuator pool was added in varying concentrations toeight separate reactions, each containing pairs of UD and DD oligos todetect 443 target sequences, among them GAPDH_2, while the concentrationof UD and DD were kept constant. The result for ligation productsindicating the presence of GAPDH_2 are shown:

concentration of added bridge attenuator average number relative to(relative to UD) of reads positive control no bridge added 4478 (100.0%)+1X bridge 2438 54.4% +3X bridge 1429 31.9% +10X bridge  602 13.5%

As shown, inclusion of bridge attenuators reduces the number of productsdetected for target sequence GAPDH_2 in a dose-dependent fashion.Moreover, FIG. 6 shows that for selected target sequences, adding thebridge attenuators for GAPDH_2 allowed the lower-abundance targetsequences to be detected at higher, but representative levels.

Skilled artisans will appreciate that additional embodiments are withinthe scope of the invention. The invention is defined only by thefollowing claims, and limitations from the specification or its examplesshould not be imported into the claims.

We claim:
 1. A set of attenuator oligonucleotides for a sample type thathas a plurality of relatively high abundance target sequences (HATs),each HAT having a downstream region (DR) and an adjacent upstream region(UR), wherein the set comprises for each HAT: (a) a downstream detectoroligo (DDO) comprising a P1 primer sequence and a region complementaryto the downstream region of the HAT target sequence (DR′), and (b) anupstream detector oligo (UDO) comprising a region complementary to theupstream region of the HAT target sequence (UR′) and a P2′ primerhybridization sequence, wherein both the downstream detector and theupstream detector are capable of hybridizing simultaneously to the HATtarget sequence under predetermined hybridization conditions, and; (c)an attenuator oligo comprising a portion of the DR′ or the UR′ ofsufficient length to specifically bind to a corresponding portion of theDR or UR of the HAT target sequence, and further comprising a sequence Xthat is different from P1 or a sequence Y that is different from P2′. 2.The set of attenuator oligonucleotides of claim 1, further comprising apool of primer oligos comprising a P1 sequence and a pool of primeroligos comprising a P2 sequence.
 3. The set of attenuatoroligonucleotides of claim 1, wherein an attenuator comprises a portionat least partially complementary to one of the DR or UR of a HAT, andfurther comprises a portion at least partially complementary to theother region of the HAT.
 4. The set of attenuator oligonucleotides ofclaim 1, wherein an attenuator comprises a portion that is complementaryto at least a portion of the DR of a HAT and another portion that iscomplementary to at least a portion of the UR of the HAT.
 5. The set ofattenuator oligonucleotides of claim 1, wherein an attenuator comprisesat least one mismatched base relative to the DR or UR of a HAT.
 6. Theset of attenuator oligonucleotides of claim 1, wherein an attenuatorcomprises a portion partially complementary to the DR of a HAT, andcomprises 1, 2, or 3 mismatched bases at any of the positions at the 3′end or at 1 or 2 positions from the 3′ end.
 7. The set of attenuatoroligonucleotides of claim 1, wherein an attenuator comprises a portionpartially complementary to the UR of a HAT, and comprises 1, 2, or 3mismatched bases at any of the positions at the 5′ end or at 1 or 2positions from the 5′ end.
 8. The set of attenuator oligonucleotides ofclaim 1, wherein, under the hybridization conditions in (b), thesequence X does not specifically bind to the P1 sequence or itscomplement; or the sequence Y does not specifically bind to the P2sequence or its complement.
 9. The set of attenuator oligonucleotides ofclaim 1, wherein an attenuator comprises a nonamplifiable segmentcomprising a reverse complementary sequence.
 10. The set of attenuatoroligonucleotides of claim 1, wherein the DR of a HAT is connected to theUR of the HAT by a flexible linker.
 11. The set of attenuatoroligonucleotides of claim 1, wherein an attenuator is phosphorylated.12. The set of attenuator oligonucleotides of claim 1, wherein anattenuator is not phosphorylated.
 13. The set of attenuatoroligonucleotides of claim 1, wherein an attenuator further comprises aportion that is complementary to a region adjacent to the DR or UR of aHAT.
 14. The set of attenuator oligonucleotides of claim 1, wherein theDR and UR of a HAT are separated by one or more nucleotides, and anattenuator has a portion that is complementary to the one or morenucleotides.
 15. The set of attenuator oligonucleotides of claim 1,wherein an attenuator comprises a selectively cleavable site.
 16. Theset of attenuator oligonucleotides of claim 1, wherein an attenuatorfurther comprises a nonextendable or nonligatable blocking group. 17.The set of attenuator oligonucleotides of claim 1, wherein an attenuatoris detectably labeled.
 18. The set of attenuator oligonucleotides ofclaim 1, wherein an attenuator comprises a barcode sequence.
 19. The setof attenuator oligonucleotides of claim 18, wherein the barcode sequenceis specific to a HAT.
 20. The set of attenuator oligonucleotides ofclaim 1, wherein a HAT comprises a sequence partially encoding a productselected from the group consisting of cytochrome c, glyceraldehyde3-phosphate dehydrogenase (GAPDH), ribosomal protein L7 (RPL7),ribosomal protein S6 (rpS6), snRNA RNUs, phosphoglycerokinase (PGK),tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation proteinzeta (YWHAZ), beta-actin, and beta-tubulin.
 21. The set of attenuatoroligonucleotides of claim 1, wherein the attenuator comprises a portionof the DR′ and a portion of the UR′.
 22. The set of attenuatoroligonucleotides of claim 9, wherein the reverse complementary sequenceforms a hairpin loop under the hybridization conditions of (b).